Development of polymer lipid particle technology for drug discovery

Kelly, Bethan Sian (2025). Development of polymer lipid particle technology for drug discovery. University of Birmingham. Ph.D.

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Abstract

G Protein-Coupled Receptors (GPCRs) represent 30-40% of all drug targets and have a massive therapeutic potential. Investigations into GPCR structure and function are often limited by their instability, flexible structure and location within the plasma membrane. Improved methodology to produce stable soluble GPCRs in an environment like the plasma membrane would be impactful for drug discovery. Amphipathic polymers, such as poly(styrene-co- maleic acid) (SMA), can be used as a solubilisation agent to produce polymer lipid particles (PoLiPa). These nanodiscs can encapsulate membrane proteins and surrounding lipids. PoLiPa have been shown to have a stabilising effect lending itself to GPCR investigations.

This project shows the expression, solubilisation and purification of GPCRs using PoLiPa technology, and preliminary data showing evidence that GPCRs are functional in an SMA lipid particle (SMALP). Firstly, the production of soluble human Neurotensin Receptor (hNTR1), expressed in E. coli, with a wild-type sequence was proven possible utilising SMA but low expression prevented its purification. Secondly, two MBP-fused GPCRs (rat NTR1 and Cannabinoid Receptor 2 (CB2)), expressed in E. coli, were solubilised and purified using SMA and were resistant to unfolding. Thirdly, mammalian cell expressed GPCRs (hNTR1 and CB2) were solubilised with different polymers and CB2 was successfully purified. Finally, SMALP-encapsulated GPCRs were proven suitable for functional analysis by fluorescent tryptophan spectroscopy (FTS), fluorescent correlation spectroscopy (FCS) and spectral shift assays. Key findings include the preferential solubilisation of GPCR oligomers or glycosylation forms dependent on the polymer type and hydrophobicity; the importance of resin types and excess polymer concentration on purification yield and allosteric modulation of Mini-G proteins for GPCR ligand binding.

It is anticipated that this project will be a starting point for other GPCR investigations to optimise the use of PoLiPa technology. This project presents key hurdles in the GPCR production process and a toolkit to overcome issues such as low expression, instability, and purification contamination.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):